Compound I Formation and Reactivity in Dimeric Chlorite Dismutase: Impact of pH and the Dynamics of the Catalytic Arginine

The heme enzyme chlorite dismutase (Cld) catalyzes the degradation of chlorite to chloride and dioxygen. Many questions about the molecular reaction mechanism of this iron protein have remained unanswered, including the electronic nature of the catalytically relevant oxoiron(IV) intermediate and its interaction with the distal, flexible, and catalytically active arginine. Here, we have investigated the dimeric Cld from Cyanothece sp. PCC7425 (CCld) and two variants having the catalytic arginine R127 (i) hydrogen-bonded to glutamine Q74 (wild-type CCld), (ii) arrested in a salt bridge with a glutamate (Q74E), or (iii) being fully flexible (Q74V). Presented stopped-flow spectroscopic studies demonstrate the initial and transient appearance of Compound I in the reaction between CCld and chlorite at pH 5.0 and 7.0 and the dominance of spectral features of an oxoiron(IV) species (418, 528, and 551 nm) during most of the chlorite degradation period at neutral and alkaline pH. Arresting the R127 in a salt bridge delays chlorite decomposition, whereas increased flexibility accelerates the reaction. The dynamics of R127 does not affect the formation of Compound I mediated by hypochlorite but has an influence on Compound I stability, which decreases rapidly with increasing pH. The decrease in activity is accompanied by the formation of protein-based amino acid radicals. Compound I is demonstrated to oxidize iodide, chlorite, and serotonin but not hypochlorite. Serotonin is able to dampen oxidative damage and inactivation of CCld at neutral and alkaline pH. Presented data are discussed with respect to the molecular mechanism of Cld and the pronounced pH dependence of chlorite degradation.

and S3: Reaction of wild-type CCld and the variants Q74V and Q74E with ClO 2at pH 5.0 and pH 9.0 S9 Figure S4: Calibration of the rapid freeze-quench device from BioLogic and CW X-band EPR spectrum of wild-type CCld in presence of a 300-fold molar excess of chlorite S11 Figure S5: Interconversion of spectral features of wild-type CCld and the variants Q74V and Q74E mediated by hypochlorite at pH 5.0 S12 Figure S6: Kinetics of Compound I formation of the CCld variants Q74V and Q74E at pH 5.0, 7.0 and 9.0 S13 Figure S7: CW X-band EPR spectrum of wild-type CCld in presence of a 10-fold molar excess of hypochlorite at pH 5.0 S14 Figure S8: CW X-band EPR spectra of wild-type CCld in presence of a 10-fold molar excess of hypochlorite at pH 5.0 obtained by RFQ and N 2 flash-freezing S15 Figure S9: Reaction of wild-type CCld Compound I with iodide at pH 5.0 S16 Figure S10: Reaction of wild-type CCld Compound I with chlorite at pH 5.0 and 7.0 S17 Figure S11: Impact of serotonin on chlorite degradation and O 2 production at pH 7.0 S18 Figure S12, S13 and S14: Reaction of wild-type CCld and the variants Q74V and Q74E with ClO 2in the presence of serotonin at pH 5.0, pH 7.0 and pH 9 S19 system with S = 5/2) to the azide-bound low-spin (S = 1/2) form. 1 N 3 -+ Mb(High-Spin) → Mb(Low-Spin)-N 3 [Eq. 1] In this work we adapted the method decribed by Pievo et. al. 2 to our system. More specifically, solutions of myoglobin from horse skeletal muscle (Sigma Aldrich) and sodium azide were prepared at a concentration of 1 mM and 5 mM, respectively, in order to work with a 5-fold molar excess of azide with respect to myoglobin. Lyophilized myoglobin was dissolved directly in 100 mM Tris-HCl buffer, pH 7. Sodium azide salt was dissolved in MilliQ water.
Myoglobin and azide solutions were loaded on the freeze-quench syringes and samples at different time points were collected. The freezing bath consisted of cold isopentane at a temperature of -122  3 °C. For each time point, 3 replicates were collected by mixing 70 μL of the myoglobin solution with 70 μL of the azide solution (final concentrations of myoglobin and azide being 0.5 mM and 2.5 mM, respectively).
The relative amounts of high-spin and low-spin myoglobin were assessed by CW Xband EPR spectroscopy. Measurements were performed at 9.95  0.05 K under non-saturating conditions. The obtained spectra are plotted in Figure S4A, while the results of the calibration calculation are depicted in Figure S4B, where the X-axis represents the apparent reaction time The parameter λ was calculated by measuring the CW X-band EPR spectra of a sample of Mb at pH 7.0 in absence of azide (resting state, 100 % high-spin) and a sample of Mb in presence of a 5-fold molar excess of NaN 3 which was left reacting for 5 seconds and then flash frozen in liquid nitrogen (reacted species, 100 % low-spin). The relative intensities were calculated, for each time points and for the two extremes of the reaction, as the ratio between the peak-to-peak distance of the low-field feature of high-spin Mb and the peak-to-peak distance of the g y feature of low-spin Mb-N 3 , all taken as absolute positive values. The intercept on the X-axis gives an apparent negative time which corresponds to the freezing time, a variable difficult to estimate otherwise, since it depends on several physical factors including the heat transfer coefficients between the sample and the cryogenic bath as well as the sample particles size and shape (and in turn the extension of the surface in contact with cryogenic liquid). 2 In S5 conclusion, to obtain the real quenching time of the reaction, the freezing time obtained by the calibration has to be added to the apparent reaction time. In our setup, we estimated a freezing time of about ~ 50 ms.

Density functional theory (DFT)
Spin-unrestricted density functional theory (DFT) calculations were performed using the ORCA package. 3,4 To mimic the solvent effect the COSMO model for water was used. 5 For the geometry optimizations of ClO 2 • , the Becke-Perdew density functional (BP86) 6,7 was used.
The Ahlrichs split-valence plus polarization (SVP) basis set was used for O atoms. 8 The Ahlrich (2df,2pd) polarization functions were obtained from the TurboMole basis set library as implemented in ORCA. For the Cl atom the doubly polarized triple-zeta (TZVPP) (Ahlrichs, unpublished) basis set was used. The energy was converged to 1 × 10 − 8 Hartree (Eh) and the tolerances of convergence in the geometry optimization were 3 × 10 − 4 Eh/Bohr for the gradient and 5 × 10 − 6 Eh for the total energy. For the single point calculations of the EPR parameters of the radical, d the PBE0 functional 9 was used in combination EPR-II 10 for oxygen and TZVPP for chlorine.
(2) Pievo, R., Angerstein, B., Fielding, A. J., Koch, C., Feussner, I., and Bennati, M. (2013) A rapid freeze-quench setup for multi-frequency EPR spectroscopy of enzymatic reactions.  Tables   Table S1. Comparison between the DFT-computed g and 35 Cl hyperfine values for ClO 2 • (see details of computation in the supporting Materials and Methods) and the parameters used to simulate the EPR spectrum in Figure S4D. The experimental error is 1 on the last digit.